Difference between revisions of "Libraries, Vectors and Screening"

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=Echinoderm protocols=
+
=An index of general molecular biology techniques used in studies of ''S. purpuratus''=
  
__TOC__
 
  
 +
[[array|Arrayed Filter Manual]]<br />
 +
The techniques used to hybridize probes to arrays of cDNAs on filters are detailed here. The manual is based on pages from the RZPD with modifications installed at Caltech.
  
== '''''Books for Xenopus Research and Protocols''''' ==
+
[[download/DIGscreen.pdf|Non-radioactive library screening protocol]]<br />
*'''Normal Table of Xenopus Laevis (Daudin)''', Nieuwkoop and Faber (Third edition), 1994 [http://www.xenbase.org/literature/book.do?method=display&bookId=1]
+
The availability of sensitive chemiluminescent detection systems for hybridization screening of arrayed library filters now makes it possible to reach outcomes similar to methods using 32P labeled probes. We have used a digoxygenin based protocol with good success. It is detailed [[download/DIGscreen.pdf|here]].
**a systematic and chronological description of Xenopus laevis development
 
*'''Early Development of Xenopus laevis: A Laboratory Manual''', Sive, Grainger, and Harland, 2000 [http://www.xenbase.org/literature/book.do?method=display&bookId=22]
 
**comprehensive collection of protocols for the study of early development in Xenopus embryos
 
*'''The Laboratory Xenopus sp.''', Green, 2010 [http://www.xenbase.org/literature/book.do?method=display&bookId=222]
 
**a highly detailed manual containing Xenopus husbandry, management, veterinary care, and frog and equipment vendor information
 
*'''Xenopus Protocols: Cell Biology and Signal Transduction''', Liu (First edition), 2006 [http://www.xenbase.org/literature/book.do?method=display&bookId=206]
 
**step-by-step laboratory instructions, an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents
 
*'''Xenopus Protocols: Post-Genomic Approaches. ''', Hoppler and Vize [http://xenbase.org/literature/book.do?method=display&bookId=262]
 
**expanded second edition with novel approaches inspired by X. tropicalis genome sequencing
 
*'''Xenopus laevis: Practical Uses in Cell and Molecular Biology, Volume 36''', Kay and Peng, 1991 [http://www.xenbase.org/literature/book.do?method=display&bookId=204]
 
**detailed description of a wide range of uses for Xenopus laevis oocytes and embryos in cell and molecular biology
 
*'''Transgenic Xenopus: Microinjection Methods and Developmental Neurobiology''', Seidman and Soreq, 1997 [http://www.xenbase.org/literature/book.do?method=display&bookId=203]
 
**referenced guide to the use of microinjected embryos studying the role and regulation of nervous system proteins during development
 
*'''Atlas of Xenopus Development''', Bernardini, Prati, Bonetti, and Scari, 1999 [http://www.xenbase.org/literature/book.do?method=display&bookId=202]
 
**scanning, transmission, and light microscopy images of Xenopus embryonic development
 
*'''The early development of Xenopus laevis. An atlas of the histology''', Hausen and Riebesell, 1991 [http://www.xenbase.org/literature/book.do?method=display&bookId=124]
 
**detailed histological sections of Xenopus embryonic development
 
*'''Color Atlas of Xenopus laevis Histology''', Wiechmann and Wirsig-Wiechmann, 2003 [http://www.xenbase.org/literature/book.do?method=display&bookId=82]
 
**central source on the microscopic anatomy of cells, tissues, and major organs of Xenopus laevis
 
  
 +
[[subtrmethods|Subtractive Probe Analysis]]<br />
 +
Our techniques for complex probes to be used on arrayed filters for gene discovery have been perfected by Jonathan Rast in the Davidson Laboratory. The investigation underlying this work is published: Rast J. P. et al, Developmental Biology 228 (2): 270-286, 2000. Supplementary data is presented here, as well.
  
== '''''Online Xenopus Resources''''' ==
+
[[libprepd|Additional Notes on cDNA Library Preparations]]<br />
 +
The procedures used to construct the cDNA libraries are described here.
  
=== '''External links to the Journal of Visualized Experiments (JOVE) - Xenopus embryo''' [http://www.jove.com/keyword/xenopus+embryo] - excellent video demonstrations ===
+
[[epgfp|The Reporter Plasmid, EpGFPII]]<br />
**Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles - Terni et al. -[https://www.jove.com/video/58028/functional-evaluation-of-olfactory-pathways-in-living-xenopus-tadpoles]
+
The sequence and map of the reporter construct described in: R. A. Cameron, P. Oliveri, J. Wyllie, and E. H. Davidson (2004) cis-Regulatory Activity of Randomly Chosen Genomic Fragments from the Sea Urchin. Gene Expression Patterns 4, 205-213 can be found here.
**Obtaining Eggs from Xenopus laevis Females - Cross & Powers [http://www.jove.com/video/890/obtaining-eggs-from-xenopus-laevis-females]
 
**Microinjection of Xenopus Laevis Oocytes - Cohen et al. - [http://www.jove.com/video/1106/microinjection-of-xenopus-laevis-oocytes]
 
**Fertilization of Xenopus oocytes, Host Transfer Method - Schneider et al. [http://www.jove.com/video/1864/fertilization-of-xenopus-oocytes-using-the-host-transfer-method]
 
**Tissue Determination Using Animal Cap Transplant (ACT) Assay in X. laevis - Viczian & Zuber [http://www.jove.com/video/1932/tissue-determination-using-animal-cap-transplant-act-assay-xenopus]
 
**Blastomere Explants to Test for Cell Fate Commitment - Grant et al. [http://www.jove.com/video/4458/blastomere-explants-to-test-for-cell-fate-commitment-during-embryonic]
 
**Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation - Amaya & Kroll [http://www.jove.com/video/2010/production-transgenic-xenopus-laevis-restriction-enzyme-mediated]
 
**Organizer and Animal Pole Explants from X. laevis Embryos and Cell Adhesion Assay - Ogata & Cho [http://www.jove.com/video/187/dissection-organizer-animal-pole-explants-from-xenopus-laevis-embryos]
 
**Plastic Embedding and Sectioning of X. laevis Embryos - Ogata1 et al. [http://www.jove.com/video/188/plastic-embedding-and-sectioning-of-xenopus-laevis-embryos]
 
**Visualizing RNA Localization in Xenopus Oocytes - Gagnon & Mowry [http://www.jove.com/video/1704/visualizing-rna-localization-in-xenopus-oocytes]
 
**Neural Explant Cultures from Xenopus laevis - Lowery et al. [http://www.jove.com/video/4232/neural-explant-cultures-from-xenopus-laevis]
 
**Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling - Itoh & Sokol [http://www.jove.com/video/2700/polarized-translocation-fluorescent-proteins-xenopus-ectoderm]
 
**Dissection, Culture, and Analysis of X. laevis Embryonic Retinal Tissue - McDonough et al. [http://www.jove.com/video/4377/dissection-culture-analysis-xenopus-laevis-embryonic-retinal]
 
**X. tropicalis Egg Extracts to Identify Microtubule-associated RNAs - Sharp & Blower [http://www.jove.com/video/50434/production-xenopus-tropicalis-egg-extracts-to-identify-microtubule]
 
**Electroporation of Craniofacial Mesenchyme - Tabler & Liu [http://www.jove.com/video/3381/electroporation-of-craniofacial-mesenchyme]
 
**Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures - Yazejian et al. [http://www.jove.com/video/50253/simultaneous-pre-post-synaptic-electrophysiological-recording-from]
 
**Single Cell Electroporation in vivo within the Intact Developing Brain - Hewapathirane & Haas - [http://www.jove.com/video/705/single-cell-electroporation-in-vivo-within-the-intact-developing-brain]
 
**Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells - Joshi & Davidson [http://www.jove.com/video/1949/live-cell-imaging-quantitative-analysis-embryonic-epithelial-cells]
 
**Preparation and Fractionation of Xenopus laevis Egg Extracts - Cross & Powers [http://www.jove.com/video/891/preparation-and-fractionation-of-xenopus-laevis-egg-extracts]
 
**In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts - Cross & Powers [http://www.jove.com/video/908/in-vitro-nuclear-assembly-using-fractionated-xenopus-egg-extracts]
 
**Study of the DNA Damage Checkpoint using Xenopus Egg Extracts - Willis et al. [http://www.jove.com/video/4449/study-of-the-dna-damage-checkpoint-using-xenopus-egg-extracts]
 
**Two Types of Assays for Detecting Frog Sperm Chemoattraction - Burnett et al. [http://www.jove.com/video/3407/two-types-of-assays-for-detecting-frog-sperm-chemoattraction]
 
**Comparative in vivo Study of gp96 Adjuvanticity in X. laevis - Nedelkovska et al. [http://www.jove.com/video/2026/comparative-vivo-study-gp96-adjuvanticity-frog-xenopus]
 
**Patch Clamp and Perfusion Techniques - Yang et al. [http://www.jove.com/video/2269/patch-clamp-perfusion-techniques-for-studying-ion-channels-expressed]
 
**Patch Clamp Recording of Ion Channels - Brown et al. [http://www.jove.com/video/936/patch-clamp-recording-of-ion-channels-expressed-in-xenopus-oocytes]
 
**Cation Transport in Xenopus Oocytes - Dürr et al.[http://www.jove.com/video/50201/measuring-cation-transport-nak-hk-atpase-xenopus-oocytes-atomic]
 
**Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo - BC.Durand [https://www.jove.com/video/53474/stem-cell-like-xenopus-embryonic-explants-to-study-early-neural]
 
**Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation - Millet & Monsoro-Burq [https://www.jove.com/video/51118/dissection-xenopus-laevis-neural-crest-for-vitro-explant-culture-or]
 
  
=== '''External links to Cold Spring Harbor Xenopus Protocols''' [http://cshprotocols.cshlp.org/search?fulltext=Xenopus&submit=yes&x=18&y=12&tocsectionid=protocol&tocsectionid=recipe&tocsectionid=topic+introduction&tocsectionid=emerging+model+organisms&tocsectionid=product+protocol&tocsectionid=kit&tocsectionid=information+panel] - subscription required, select articles listed ===
+
[[bac_vector_library|BAC Library Vector]]<br />
**Housing and Feeding of Xenopus laevis - Sive et al. [http://cshprotocols.cshlp.org/content/2007/5/pdb.top8.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
+
The BAC vector pBACe3.6 is the one we have used in the preparation of all of our macro-array genomic libraries. Constructed in Pieter J. de Jong's laboratory at the Children's Hospital Oakland Research Center, it has been used extensively for genomic libraries and sequencing projects of many species.
**Inducing Ovulation in Xenopus laevis - Sive et al. [http://cshprotocols.cshlp.org/content/2007/5/pdb.prot4734.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Xenopus laevis In Vitro Fertilization and Natural Mating Methods - Sive et al. [http://cshprotocols.cshlp.org/content/2007/5/pdb.prot4737.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Egg Collection and In Vitro Fertilization of the Western Clawed Frog Xenopus tropicalis - Showell & Conlon [http://cshprotocols.cshlp.org/content/2009/9/pdb.prot5293.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Isolation of Xenopus Oocytes - Sive et al. [http://cshprotocols.cshlp.org/content/2010/12/pdb.prot5534.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Isolating Xenopus laevis Testes - Sive et al. [http://cshprotocols.cshlp.org/content/2007/5/pdb.prot4735.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Dejellying Xenopus laevis Embryos  - Sive et al. [http://cshprotocols.cshlp.org/content/2007/5/pdb.prot4731.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Removing the Vitelline Membrane from Xenopus laevis Embryos - Sive et al. [http://cshprotocols.cshlp.org/content/2007/5/pdb.prot4732.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Microinjection of Xenopus Embryos - Sive et al. [http://cshprotocols.cshlp.org/content/2010/12/pdb.ip81.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Defolliculation of Xenopus Oocytes - Sive et al. [http://cshprotocols.cshlp.org/content/2010/12/pdb.prot5535.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Microinjection of Xenopus Oocytes - Sive et al. [http://cshprotocols.cshlp.org/content/2010/12/pdb.prot5536.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Animal Cap Isolation from Xenopus laevis - Sive et al. [http://cshprotocols.cshlp.org/content/2007/6/pdb.prot4744.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Xenopus laevis Keller Explants - Sive et al. [http://cshprotocols.cshlp.org/content/2007/6/pdb.prot4749.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Microinjection of RNA and Preparation of Secreted Proteins from Xenopus Oocytes - Sive et al. [http://cshprotocols.cshlp.org/content/2010/12/pdb.prot5538.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Calibration of the Injection Volume for Microinjection of Xenopus Oocytes and Embryos - Sive et al. [http://cshprotocols.cshlp.org/content/2010/12/pdb.prot5537.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Isolation of DNA from Red Blood Cells in Xenopus - Sive et al. [http://cshprotocols.cshlp.org/content/2012/1/pdb.prot067462.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Investigating Morphogenesis in Xenopus Embryos: Imaging Strategies, Processing, and Analysis - Kim & Davidson [http://cshprotocols.cshlp.org/content/2013/4/pdb.top073890.abstract?sid=47d75b27-9727-4811-8a78-0330a022b90f]
 
**Low-Magnification Live Imaging of Xenopus Embryos for Cell and Developmental Biology - Wallingford [http://cshprotocols.cshlp.org/content/2010/5/pdb.prot5425.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**High-Magnification In Vivo Imaging of Xenopus Embryos for Cell and Developmental Biology - Keiserman et al. [http://cshprotocols.cshlp.org/content/2010/5/pdb.prot5427.abstract?sid=3a8d778b-0e2c-4925-bb88-c7ade32e7bff]
 
**Preparation of Fixed Xenopus Embryos for Confocal Imaging - Wallingford [http://cshprotocols.cshlp.org/content/2010/5/pdb.prot5426.abstract?sid=3a8d778b-0e2c-4925-bb88-c7ade32e7bff]
 
**Whole-Mount Fluorescence Immunocytochemistry on Xenopus Embryos - Lee et al. [http://cshprotocols.cshlp.org/content/2008/2/pdb.prot4957.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Generation of Transgenic Xenopus laevis - Kroll & Amaya - [http://cshprotocols.cshlp.org/content/2007/9/pdb.prot4838.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae] [http://cshprotocols.cshlp.org/content/2007/9/pdb.prot4839.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae] [http://cshprotocols.cshlp.org/content/2007/9/pdb.prot4840.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**In Vivo Time-Lapse Imaging of Neuronal Development in Xenopus - Ruthazer et al. [http://cshprotocols.cshlp.org/content/2013/9/pdb.top077156.abstract?sid=47d75b27-9727-4811-8a78-0330a022b90f]
 
**Photoconversion for Tracking the Dynamics of Cell Movement in Xenopus laevis Embryos - Chernet et al. [http://cshprotocols.cshlp.org/content/2012/6/pdb.prot068502.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Single-Cell Electroporation in Xenopus - Liu & Haas [http://cshprotocols.cshlp.org/content/2011/9/pdb.top065607.abstract?sid=009b88bf-7bfb-4279-9528-70643a25bbae]
 
**Imaging Axon Pathfinding in Xenopus In Vivo - Leung & Holt [http://cshprotocols.cshlp.org/content/2012/9/pdb.prot070003.abstract?sid=47d75b27-9727-4811-8a78-0330a022b90f]
 
**A Versatile Protocol for mRNA Electroporation of Xenopus laevis Embryos - Chernet & Levin [http://cshprotocols.cshlp.org/content/2012/4/pdb.prot067694.abstract?sid=47d75b27-9727-4811-8a78-0330a022b90f]
 
  
 +
[[download/Lab_Manual.pdf|Sea Urchin Fertilization Laboratory]]<br />
 +
Vic Vacquier developed a handout for university students on how to observe sea urchin fertilization in a university lab class setting. All that is needed are sea urchin gametes, a microscope, a few solutions and this handout. The handout works well for both 1 or 2 three hour labs. The student reads through the handout, makes the simple observations, and does the simple, direct experiments to gain insight into the fascinating process of sperm-egg interaction and egg activation.
  
== '''''General Research Protocols''''' ==
+
[http://www.ncbi.nlm.nih.gov/pubmed/?term=23124201 Vectors for generation of exogenous mRNAs fused to fluorescent proteins for expression in urchins]<br />
 
+
The expression of exogenous proteins in sea urchins embryos, by injection of recombinant mRNAs, is routine procedure. The Hamdoun lab has generated a vector set with the major color and tag position variants that is useful for fluorescent protein tagging and expression in urchins. The complete vector set and can be obtained here: https://www.addgene.org/fluorescent_proteins/hamdoun/. A description of the vectors can be found in Gokirmak et al., 2012, JBC 287:43876-83. PMID: 23124201
=== '''''Animal Husbandry''''' ===
 
*obtain frogs and frog housing equipment [http://www.xenbase.org/other/obtain.do]
 
*Xenopus Express links [http://www.xenopus.com/links.htm] - Xenopus Express husbandry [http://www.xenopus.com/husbandry.htm]
 
*[[Xenopus tropicalis and Xenopus laevis environmental parameter standard operating procedure (Kroll lab)]]
 
 
 
 
 
=== '''''[[Lab Solutions and Reagents]]''''' (click each to view expanded content) ===
 
*[[culture media]]
 
*[[immunohistochemistry]]
 
*[[in situ hybridization]]
 
*[[fixatives]]
 
*[[DNA/RNA preparation]]
 
*[[embryo manipulation/dissection]]
 
*[[microinjection]]
 
*[[general buffers and salt solutions]]
 
 
 
 
 
=== '''''Generating Embryos''''' ===
 
*[[inducing egg laying via hCG injection (Vize lab)]]
 
*[[anesthesia, in vitro fertilization, and natural mating of Xenopus tropicalis (Conlon lab)]]
 
*[[benzocaine stock, 100x (Khokha lab)]]
 
*[[in vitro fertilization of Xenopus laevis (Conlon lab)]]
 
*[[high-salt solution for egg laying (Zorn lab/Ron Laskey)]]
 
*[[dejellying embryos (Sive et al. 2000)]]
 
*[[dejellying embryos (Zorn lab)]]
 
 
 
 
 
=== '''''Transgenesis''''' ===
 
*[[transgenesis in Xenopus laevis - adapted from Amaya et al 2001 (Conlon lab)]]
 
*[[tadpole tail sampling for genomic DNA (Conlon lab)]]
 
*''Production of transgenic Xenopus laevis by restriction enzyme mediated integration and nuclear transplantation.'' Amaya E, Kroll K.  J Vis Exp. 2010 Aug 21;(42). [http://www.jove.com/video/2010/production-of-transgenic-xenopus-laevis-by-restriction-enzyme-mediated-integration-and-nuclear-transplantation]
 
*''A method for generating transgenic frog embryos.'' Ishibashi S, Kroll KL, Amaya E.  Methods Mol Biol. 2008;461:447-66.  [http://www.springerlink.com/content/vn5v1470g87620t1/#section=96225&page=1]
 
*''A method for generating transgenic frog embryos.'' Amaya E, Kroll KL.  Methods Mol Biol. 1999;97:393-414. [http://www.springerlink.com/content/vn5v1470g87620t1/#section=96225&page=1]
 
 
 
 
 
=== '''''in situ Hybridization''''' ===
 
*[[in situ hybridization, brief version (Harland lab)]]
 
*[[whole-mount in situ hybridization (Conlon lab)]]
 
**[[in situ checklist (Conlon lab)]]
 
*[[whole-mount in situ hybridization - LNA probes (Wheeler lab)]]
 
*[[fluorescent in-situs and FCIS (Vize lab)]]
 
**[[Wholemount embryo FISH / Double FISH]]
 
**[[Flourescin Tyramide Synthesis]]
 
**[[Cy3 Tyramide Synthesis]]
 
**[[FCIS]]
 
*[[RNA Whole-Mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA) (Myers and Scarlett labs)]]
 
 
 
 
 
=== '''''Immunohistochemistry''''' ===
 
*[[wholemount antibody staining (CSH Protocols) (Wallingford lab)]]
 
*[[wholemount antibody staining - alkaline phosphatase detection (Conlon lab)]]
 
*[[wholemount antibody staining - adapted from Dan Weeks' protocol (Conlon lab)]]
 
*[[immunostaining sectioned embryos (Conlon lab)]]
 
*[[hydrochloric acid antigen retrieval (Zorn lab)]]
 
*[[RNA Whole-Mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA) (Myers and Scarlett labs)]]
 
 
 
 
 
=== '''''ChIP protocols''''' ===
 
*'''Chromatin immunoprecipitation analysis of Xenopus embryos.''', Methods Mol Biol. 2012;917:279-92. [http://www.xenbase.org/literature/article.do?method=display&articleId=45851] [http://www.ncbi.nlm.nih.gov/pubmed/22956095]
 
**Akkers RC, Jacobi UG, Veenstra GJ.
 
*'''Chromatin immunoprecipitation in early Xenopus laevis embryos.''', Dev Dyn. 2009 Jun;238(6):1422-32. [http://www.xenbase.org/literature/article.do?method=display&articleId=39423] [http://www.ncbi.nlm.nih.gov/pubmed/19334278]
 
**Blythe SA, Reid CD, Kessler DS, Klein PS.
 
 
 
 
 
=== '''''Histology''''' ===
 
*[[cryosectioning Xenopus embryos (Conlon lab)]]
 
 
 
 
 
=== '''''Embryo Staining Protocols (non in situ)''''' ===
 
*[[benzidine-peroxidase staining for blood (Conlon lab)]]
 
*[[benzidine-peroxidase staining for blood (Thomsen lab)]]
 
*[[apoptosis via TUNEL (Veenstra lab)]]
 
*[[TUNEL staining of whole Xenopus embryos - Harland protocol (Conlon lab)]]
 
 
 
 
 
=== '''''Immuno and Protein Protocols''''' ===
 
*[[immunoprecipitation (Stukenberg lab)]]
 
*[[immunofluorescence with biotinylated antibodies (Stukenberg lab)]]
 
*Purifying Polyclonal Antibodies using Affinity Columns/Chromatography
 
*[[Making an Affinity Column]]
 
*[[Running an Affinity Column]]
 
*[[whole embryo protein extraction with freon (Conlon lab)]]
 
*[[SDS-PAGE gels (Conlon lab)]]
 
*[[western blot protocol (Conlon lab)]]
 
*[[silver staining protein gels (Stukenberg lab)]]
 
 
 
 
 
=== '''''Nucleic Acid Protocols''''' ===
 
*RNA Isolation
 
**[[Preparation of RNA Using Proteinase K/LiCl (Sive et al. 2000)]]
 
**[[Preparation of RNA (In Vitro Transcription) (Sive et al. 2000)]]
 
*DNA Isolation
 
**[[Extraction of DNA from Single Embryos (Sive et al. 2000)]]
 
**[[Isolation of DNA from Red Blood Cells (Sive et al. 2000)]]
 
 
 
 
 
 
 
=== '''''[[Oocyte Transfer Technique (Heasman/Wylie labs)]]''''' ===
 
*[[Part 1: Choosing Oligodeoxyribonucleotides (ODNs)]]
 
*[[Part 2: ODN Modification]]
 
*[[Part 3: Host Transfer Technique]]
 
*[[Part 4: Fertilization and Development]]
 
 
 
 
 
=== '''''Xenopus Oocyte and Egg Extracts''''' ===
 
*[[crude interphase egg extract (Shechter lab)]]
 
*[[isolation of chromatin from egg extract and histone recovery (Shechter lab)]]
 
*[[preparation of cycling egg extract (Shechter lab)]]
 
*[[cycling extracts, mike's protocol (Stukenberg lab)]]
 
*[[cycling extracts, sara's protocol (Stukenberg lab)]]
 
*[[CSF extract, mike's protocol (Stukenberg lab)]]
 
*[[CSF extract, sara's protocol (Stukenberg lab)]]
 
*[[assaying H1 kinase in extracts (Stukenberg lab)]]
 
*[[frozen egg extracts that retain spindle assembly activity (Takagi and Shimamoto, 2017)]]
 
 
 
=== '''''Xenopus Tissue Culture''''' ===
 
*[[tissue culture of Xenopus cell lines S3, XTC (Stukenberg lab)]]
 
*[[isolation of mitotic chromosomes from XTC cells (Stukenberg lab)]]
 
*[[in-vitro kinetochore-microtubule binding assays (Stukenberg lab)]]
 

Revision as of 23:49, 10 February 2020

An index of general molecular biology techniques used in studies of S. purpuratus

Arrayed Filter Manual
The techniques used to hybridize probes to arrays of cDNAs on filters are detailed here. The manual is based on pages from the RZPD with modifications installed at Caltech.

Non-radioactive library screening protocol
The availability of sensitive chemiluminescent detection systems for hybridization screening of arrayed library filters now makes it possible to reach outcomes similar to methods using 32P labeled probes. We have used a digoxygenin based protocol with good success. It is detailed here.

Subtractive Probe Analysis
Our techniques for complex probes to be used on arrayed filters for gene discovery have been perfected by Jonathan Rast in the Davidson Laboratory. The investigation underlying this work is published: Rast J. P. et al, Developmental Biology 228 (2): 270-286, 2000. Supplementary data is presented here, as well.

Additional Notes on cDNA Library Preparations
The procedures used to construct the cDNA libraries are described here.

The Reporter Plasmid, EpGFPII
The sequence and map of the reporter construct described in: R. A. Cameron, P. Oliveri, J. Wyllie, and E. H. Davidson (2004) cis-Regulatory Activity of Randomly Chosen Genomic Fragments from the Sea Urchin. Gene Expression Patterns 4, 205-213 can be found here.

BAC Library Vector
The BAC vector pBACe3.6 is the one we have used in the preparation of all of our macro-array genomic libraries. Constructed in Pieter J. de Jong's laboratory at the Children's Hospital Oakland Research Center, it has been used extensively for genomic libraries and sequencing projects of many species.

Sea Urchin Fertilization Laboratory
Vic Vacquier developed a handout for university students on how to observe sea urchin fertilization in a university lab class setting. All that is needed are sea urchin gametes, a microscope, a few solutions and this handout. The handout works well for both 1 or 2 three hour labs. The student reads through the handout, makes the simple observations, and does the simple, direct experiments to gain insight into the fascinating process of sperm-egg interaction and egg activation.

Vectors for generation of exogenous mRNAs fused to fluorescent proteins for expression in urchins
The expression of exogenous proteins in sea urchins embryos, by injection of recombinant mRNAs, is routine procedure. The Hamdoun lab has generated a vector set with the major color and tag position variants that is useful for fluorescent protein tagging and expression in urchins. The complete vector set and can be obtained here: https://www.addgene.org/fluorescent_proteins/hamdoun/. A description of the vectors can be found in Gokirmak et al., 2012, JBC 287:43876-83. PMID: 23124201